Arylacylamidase E.C., 3.5.1.13. has been isolated from various microbial organisms, such as Pseudomonas acidovorans (ATCC 15668) by J. Alt et al., in J. Gen. Microb. 87: 260 (1975) and in Eur. J. Biochem. 53: 357 (1975); Bacillus schaericus (ATCC 12123) by G. Englehart et al.; Pseudomonas fluorescens (ATCC 39005) and Pseudomonas putida (ATCC 39004) by Hammond et al., U.S. Pat. No. 4,414,327.
Arylacylamidases from different microorganisms have been used in the determination of anilides such as acetaminophen by Hammond et al. in Anal. Biochem. 143: 152 (1984) and in U.S. Pat. No. 4,999,288.
However, it has recently been estimated that a great many of all in vitro diagnostic tests conducted annually in this country are not reliable. Unreliable tests can result in unnecessary medical treatment, the withholding of unnecessary treatment, and incorrect treatment. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years, and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems in part from the fact that the exact nature of enzymes, as well as the mechanisms of their action, remain unknown for the most part. At present, the greatest limitation on the use of enzymes lies in the unstable characteristics of the enzymes themselves. Current methodologies require the use of numerous labile ingredients.
The present commercial state of the art for stabilizing the reactive ability of enzymes is by locking them into a solid matrix either by freeze drying, dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries, and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable, and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and the usefulness of the product is further limited by packaging modes and sizes. It Is far more convenient for the ultimate user to obtain the enzyme in solution, as it is in this form that the enzyme is most readily used,.
Arylacylamidases in particular are very delicate and highly susceptible to loss in activity. These enzymes are not stable in either aqueous or lyophilized form, and thus it would be particularly useful to provide a stable arylacylamidase composition which has a reasonably long shelf life at ambient temperatures.
A number of prior art workers have attempted to stabilize a variety of enzymes, among them the arylacylamidases, with varying degrees of success. For example, Modrovich, in U.S. Pat. No. 4,310,625, discloses a method for stabilizing labile enzymes by forming a solution of the enzyme in an aqueous medium containing at least 20% organic solvent in the presence of a small amount of polymer such as gelatin. The stability can be further enhanced by including from 1-18% of salts and a bacteriostatic agent to prevent degradation of the substrate, which may serve as food for bacteria, fungi and other microorganism. None of the enzymes stabilized by this method is an arylacylamidase, and the stabilizing is effected by protecting the functional group site of the enzyme.
Hammond, in U.S. Pat. No. 4,414,327, discloses a process for preserving arylacylamidases in aqueous solution by including glycerol in the solution. The glycerol is present in amounts of from 10 to 70% glycerol volume/volume. In order further to retain the activity of the enzyme, the enzyme solution is stored at low temperatures (5 to -20.degree. C.).